CA1145330A - Enkephalin analogues - Google Patents

Abstract

A B S T R A C T
Tetrapeptide amides of the formula

Description

~533~

This invention relates to peptides, to processes for their preparation and to therapeutic compositions thereof.
According to this invention there are provided compounds for the preparation of a novel peptide compound of the formula:

R Tyr - B - Gly - D - N _ RX

wherein Rl is hydrogen or methyl;
B is D-alanine, D-valine, D-norvaline, D-serine, D-methionine or D-methionine sulphoxide;
D is L-phenylalanine or N-methyl-L-phenylalanine;
R is hydrogen, alkyl Cl 5, Ar or Ar-alkyl Cl 2;
R is a group (CH2)s - X
where s is 2 4 and X is NHR9 N ~3 RllR12R13 NR9cORll NRgcOO
NR CONH2, NR9CoNRlOR 1, N(O)nRllR 2, COOR9, or oCONR9R10 (where R is hydrogen, alkyl Cl 3, Ar or ArCH2; R10 is hydrogen or alkyl Cl 3; R 1 is alkyl Cl 3, Ar or ArCH2; R is alkyl Cl 3;
R13 is alkyl Cl 3; and n is O or l); or the group 1!~

5~33~

- N ~ Rx CH2CH2 ¦ is N Q
R8 \ (CH2)m where M is 1-2 and Q is SO or N(O)R l;
Ar is phenyl or phenyl substituted by chlorine, methyl, hydroxy or methoxy; and their acid addition salts.
Other examples of B include D-a-aminobutyric acid, D-leucine, D-norleucine, D-threonine and D-methionine.

,:

~ 33~

In an aspect of the invention there are provided compounds of Formula I
wherein Rl is hydrogen, methyl, allyl, dimethylallyl, propargyl, cyclopropylmethyl, cyclobutylmethyl or phenethyl;
B is the group - NH - CR H - CO -in which yl C1-6 or CqH2qYCrH2r+l ~where q is 1-2 r is 0-1 and Y is 0, S, SO or S02), the group having the D configuration;
D is the group in which R5 is hydrogen or methyl;
lS R6 is Ar-alkyl Cl 3, the group having the L-configuration;
R8 is hydrogen or alkyl Cl 5;
Rx is a group (CH2)s - X
where s is 2-4 and X is NHR9, NR9CORll, NR9CoNH2, NR9CONRlORll, N(O)nRllR12, COOR9, CONR9R10, NR9COORll or OCONR9R10 (where R9 is hydrogen or alkyl Cl 3;
R10 is hydrogen or alkyl Cl 3; Rll is alkyl Cl 3;
R12 is alkyl Cl 3 and n is O or l);

33~:) Ar is phenyl or phenyl substituted by chlorine, methyl, hydroxy or methoxy; and their acid addition salts.
The symbols used herein for amino-acid derivatives are those customarily used in peptide chemistry such as are set out in Biochem. J. 126. 773 (1972). All amino-acid residues are of the natural or L-configuration unless specified otherwise.
The invention also provides therapeutic composi-tions comprising a compound o~ the formula, or a pharmaceutically acceptable acid addition sait thereof, in association with a pharmaceutically acceptable diluent or carrier. Suitable salts are the hydrochlorides, phosphates, citrates and acetates.
The compounds of the invention exhibit pharmacological activity. Thus for example, by in vitro testing they have been found to have affinity for opiate receptor sites and as such may be used as analgesics, narcotic antagonists or anti~diarrhoeal agents. The test used for the detection of their agonist activity at opiate receptor sites is that employing the transmurally stimulates guinea pig ileum preparation described in Kosterlitz et al., Brit. J. Pharmacol, and Chemotherapy 33. 266-276 ~ 3 3 ~

(1968). They have also been shown to exhibit analgesic activity when tested in vivo for example in the mouse phenyl-quinone anti writhing test of Hendershot et al., J. Pharmac. Exp. Ther. 125, 237 (1959) and by the rat tail flick test ~intraveneous administration) according to Jansen et al., Ar~neim Forsch. 13, 266 (1963).
The Compounds of the invention may be prepared by the standard methods of peptide chemistry.
Thus they may be produced by sequential coupling, of suitably protected and activated amino acids by either classical solution methods or solid phase procedures, or by coupling fragments consisting of suitably protected peptides.
Details concerning the selection of protecting groups and methods for their incorporation as well as suitable reaction conditions for forming amido ~peptide) linkages and removal of protecting groups may be found in the following references:
~a) Houben Weyl ~ethoden der Organischen Chemie Vol. 16 Parts I and II Syntheses von Peptiden ~Thieme 1974) , .

~533~

(b) Schroder & Lubke "The Peptides"
Academic Press (1965) Thus the compounds of formula I may be prepared by condensing a compound of formula II
Y - Ml - OH II
where Y is a suitable N-protecting group and Ml is a suitably protected amino acid or peptide residue, with a compound of formula III
H - M2 ~ N _ Rx III

~ where M2 is a suitably protected amino acid or peptide i residue ~where Ml and M2 together represent -Tyr-B-Gly-D- or a protected derivative thereof), followed by removal of the protection.
The coupling of II and III may be achieved by the standard methods of peptide synthesis, either with or without isolation of the activated component corresponding to the compound of formula II.
Conveniently a protected N-terminal dipeptide is coupled with a C-terminal dipeptide amide or a protected N-terminal tripeptide is coupled with a C-terminal amino acid amide followed in either case by the removal of the protection.

', . ~ ' ~' ' ' 53 ~ O

A useful NH-protecting group is t-bu~yl~xy carbonyl and a particularly useful OH protecting group is the t-butyl radical both of which may be removed by treatment of the protected compound with hydrogen chloride in a solvent such- 'as ,ethyl-" acetate or "' ' ' ~
acetic --- acid or''with trifluoroacetic ' acid.
The compounds of Formula I in which B is a group NH CR3H-Co- where R3 is CqH2qYCrH2r+l Y is sn or S02 may be prepared from the analogous compounds in which Y is S by oxidation with for example hydrogen peroxide in a soivent such as acetic acid or methanol. They may also be prepared 1~ by carrying out the oxidation on a protected compound of formula I folIowed by deprotection.
The compounds of Formula I in which Rx is ~CH2)S-X and X is N~O)RllR12 may also be prepared from the analogous compounds in which X is NRllR12 by oxidation with for example hydrogen peroxide in a solvent such acetic acid or methanol, or by oxidation with meta-chloroperben~oic acid. This - - :

~45331~

.~

method of oxidation is also applicable for the preparation of intermediate amino acid amides or dipeptide amides containing the group N~O)RllR12.
The compounds of Formula I in which the group R8 is N Q
\ ~CH2)m ; S and where Q is N~O)Rll, SO or SO2 may be prepared from the analogous compounds where Q is NRll or S respectively by similar methods of oxidation.
Intermediate amino acid amides or dipeptide amides containing these groups may be prepared in an analogous manner.
The invention is illustrated by the following non-limiting Examples in which temperatures are in degrees Celsius.
The following abbreviatives are used throughout BOC t-Butyloxycarbonyl But t-Butyl ~ 33V

IBCF Isobutylchloroformate Z Benzyloxycarbonyl DCCI Dicyclohexylcarbodiimide DCU Dicyclohexylurea HONSu N-Hydroxysuccinimide NMM N-~ethylmorpholine DMF Dimethylformamide DME 1,2-Dimethoxyethane THF Tetrahydrofuran 10The Yarious compounds and intermediates were : examined by thin layer chromatography (t.l.c.) on silica gel plates (Kieselgel GF 254) using the following systems:-lE methanol, chloroform 1:2 15lF methanol, chloroform 1:19 2B chloroform, methanol, acetic acid lg:9:1 3A chloroform, methanol, acetic acid, water 60:18:2:3 3B chloroform, methanol, acetic acid, water 30:18:4:6 3C chloroform, methanol, acetic acid, water 90:27:2:3 3D chloroform, methanol, acetic acid, water 30:20:2:3 S~3(~

4A isobutanol~ ethyl acetate, acetic acid, water 1:1:1:1 5B isobutanol, acetic acid, water 3:1:1 7B ethylacetate, pyridine, acetic acid, water 60:20:6:11 7C ethylacetate, pyridine, acetic acid, water lZC:20:6:11 7D ethylacetate, pyridine, acetic acid, water 240:20:6:11 7F ethylacetate, pyridine, acetic acid, water 480:20:6:11 8A chloroform, isopropanol 3:1 8B chloroform, isopropanol 9:1 EXAMPLE
L-Tyrosyl-D-alanylglycyl-L-phenylalanin-2-acetamido-ethylamide This was prepared according to the following method Tyr D-Ala Gly Phe lBOC - - OH MH2CH2CH2NHCOCH3 But I BOC _ --MHCH2CH2'~HCOOCH3 BOC--But OH Cl~H ~ _ --NHCH2CH21~HCOCH3 BOC - / ~ - NHCH2CH2MHCOCH3 H ... _ _ CH2CH2NHCOCH3 ~533(~

~1) BOC-Phe-NH_CH2CH2NHCOCH3_ BOC-Phe-OH (2.65 g) was dissolved in CH2C12 ~20 ml) and the solution cooled to -20 when NMM (1.01 g) and IBCF ~1.37 g) were added followed by H2NCH2CH2NH.COCH3 (1.2 g) after 2 minutes. The reaction mixture was allowed to attain room temperature and stirred for 21 hours when the solvent was evaporated and the gum dissolved in chloroform. The solution was washed with a saturated solution of NaHCO37 10~ aqueous citric acid, with water until neutral and finally with a saturated brine solution. The organic phase was dried (Na2S04) and evaporated to a solid which crystallised from ethyl acetate to yield the amide 1 (3.0 g).
Rf 7F 0.35.

(2) Cl H Phe-NHCH2CH2NHCOCH3_ BOC-Phe-NHCH2CH2NHCOCH3 (3 g3 was dissolved in 5M HCl in ethyl acetate (20 ml) and 2.6M HCl in acetic acid (5 ml), and stirred at ambient temper~ture for 30 minutes. The solvent was evaporated and the residue triturated several times with dry diethyl ether. The hydrochloride (2) was obtained as a hygroscopic solid (0.45 g) Rf3B 0.5 ,,. , ; . . .

53 ~

(4) BOC-Gly-Phe NHCH2CH2NHCOCH3_ This was prepared by either of the following approaches:
(a) BOC-Gly-Phe-OH ~l.O g) and H2NCH2CH2NHCOCH3 (0.4 g) were dissolved in DMF and cooled in an ice/salt bath where HONSu ~0.78 g) and DCCI (0.668 g) were added. The reaction mixture was allowed to attain room temperature and stirred overnight when the DCU was removed by filtration and the solvent evaporated. The residue was dissolved in CHC13, washed with saturated sodium bicarbonate solution (2 x 50 ml), 10~ aqueous citric acid ~2 x 50 ml) and water until neutral, and then with a saturated brine solution. The organic phase was dried (Na2S04) and evaporated. The residue was chromatographed on a silica gel column (45 cm x 2.4 cm).
The column was first eluted with CHC13/isopropanol ~19:1) then with CHC13/isopropanol (4 : 1 ). The title compound was obtained as a gum ~0.60 g) Rf8B 0.4, Rf7F 0.2 (b) BOC-Gly-OH (1.072 g) was dissolved in CH2C12 and cooled in solid C02/CCl4 bath when ~ 3 ~

NMM (0.618 g) and IBCF (0.836 g) were added.
After 2 minutes at this temperature - -Cl H .Phe-NHCH2CH2NHCOCH3 (1.75 g) and NM~I
(0.618) were added and the temperature allowed to rise to room temperature. The reaction was worked up after overnight stirring as described for (1) above. The residue was purified according to (a) above. The product was obtained as a gum (0.65 g). Rf8B 0.4, Rf7F 0.2 (5) BOC-Tyr (But)-D-Ala-Gly-Phe-NHCH2CH NHCOCH

~i) BOC-Gly-Phe-NHCH2CH2NHCOCH3 (0.64 g) was treated with 5.5M HCl in ethyl acetate (5 ml) and 2.6 M HCl in acetic acid (5 ml) at ambient temperature. A~ter 45 minutes the solvent was evaporated a~d the residue triturated with diethyl ether. The dipeptide hydrochloride was obtained as a hygroscopic solid (0.54 g) Rf 3D 0.45 (ii) BOC-Tyr(But)-D-Ala-OH (0.6 g) and Cl H Gly-Phe-NHCH2CH2NHCOCH3 (0.5 g) were dissolved in DMF (3 ml) and cooled in ice/salt bath when HONSu (0.355 g) and DCCI (0.303 g) were added followed by NMM (1.48 g in DMF, 1.5 ml). The reaction mixture was allowed to warm up to room temperature and stirred overnight. DCU was then ., i, . :
filtered off, the solvent evaporated and the residue partitioned between ethyl acetate and water. The organic layer was washed with a saturated aqueous sodium bicarbonate solution (2 x 50 ml), 10% aqueous citric acid ~2 x 50 ml), water until neutral, and finally with saturated brine solution. The organic phase was dried (Na2S04) and evaporated. The tetrapeptide was obtained as a gum (0.65 g) .
Rf7C 0.65 Rf3D 0.8.
~6) H-Tyr-D-Ala-Gly-Phe-NHCH CH NHCOCH

BOC-Tyr(But)-D-Ala-Gly-Phe-NHCH2CH2NHCOCH3 (0.65 g) was treated with excess 2.6M HCl in acetic acid for 45 minutes at ambient temperature.
The solvent was evaporated and the residue chromatographed on a silica gel column t50 cm x 2.5 cm) with solvent system 3A. The resultant gum was lyophilised to yield the title compound (0.45 g) Rf3A 0.3 ~q 22 -6.95 (c = 1, DMF) .
L-Tyrosyl-D-alanylalycyl-N-methyl-L-phenylalanine-

3-dimethylaminopropylamide i330 Tyr D-Ala Gly MePhe BOC~OH
Boc~H(cH2) 3~1e2 BOC---OH Cl H2 ~ CH2)3~e2 But BOC - . NH~CH2)3NMe2 BOC _ ~ut --OHCl H2-- ~CH2)3NMe2.HCl BOC - /_ . -NH~CH2)31~Me2 Cl H2 ¦_ _ I~H~CH2)31~Me2 This was prepared by the method of the above scheme:-~1) Boc-Mephe-NH---2cH2cH2NMe2-N-t-Butyloxycarbonyl-N-methylphenylalanine ~2.79 g) and NMM ~1.01 g) were dissolved in dichloro-methane (20 ml) and the stirred solution cooled to -20.
IBCF ~1. 37 g) was then added and the mixture stirred for S minutes before the addition of NH2CH2CH2CH2NMe2 ~1.02 g). The solution was stirred for 1 hour at -20 10 and then at room temperature for 12 hours. The mi~ture was evaporated, and the residue dissolved in ethyl .

~ - 16 -acetate ~50 ml) and an aqueous solution of sodium bicarbonate (saturated, 50 ml). The ethyl acetate layer was washed with aqueous sodium bicarbonate (saturated, 50 ml), water (2 x 50-ml) and 10~ aqueous citric acid (2 x 50 ml).- The , . .
citric acid extracts were combined, made basic with saturated sodium bicarbonate solution, and extracted with ethyl acetate (3 x 50 ml). The ethyl acetate extracts were combined, dried (Na2S04) and evaporated to yield the title compound as an oil RflE 0.24, 10 Rf3A 0.37.
(2) H-Me-Phe--NI~CH2CH2CH2NMe2 2HCl The oil from (1) above was dissolved in ethyl acetate (10 ml), and HCl in acetic acid (2.63M, 6 ml) and HCl in ethyl acetate (5.7 M, 6 ml) added. The lS solution was stirred for 2 hours and evaporated to yield the desired dihydrochloride salt ~2=2 g) as a solid RflE 0.02, Rf3A 0.03.
~3~ BOC-Gly-MePhe-NHCH2CH CH NMe2 t-Butyloxycarbonylglycine (1.05 g) and NMM
20 (0.606 g) were dissolved in dichloromethane ~12 ml) and the stirred solution cooled to -20. IBCF
(0.819 g, 6mM) was added and the mixture stirred for 5 minutes before the addition of ~2) above (2.02 g) in DMF (5 ml). The solution was stirred for 1 hour 25 at -20 and 12 hours at room temperature and then ~ 3 ~

evaporated. The residue was dissolved in ethyl acetate (50 ml) and an aqueous solution of sodium bicarbonate ~10~ 50ml). The organic phase was separated, washed with aqueous sodium bicarbonate ; 5 ~10% 3, x 50 ml) and water ~3 x 50 ml). The ethyl acetate solution was dried ~Na2S04) and evaporated to yield the title compound as an oil.
; R~lE 0.21; Rf3A 0.37 H-Gly-MePhe-NHcH2cH2cH2NMe2 2 The protected dipeptide ~3) was treated by the procedure as described for ~2) above to yield the desired dihydrochloride salt ~4) ~2.0 g) as a white solid. ~flE 0.03, Rf3A 0.03.
~5) BOC-Tyr~But)-D-Ala-Gly-MePhe-NHCH2CH2CH2NMe2_ BOC-Tyr~But)-D-Ala-OH ~1.22 g), H-Gly-MePhe-NHCH2CH2CH2NMe2.2HCl ~1.18 g), HONSu ~0.345 g) and NMM ~0.30 g) were dissoived in dry DMF ~20 ml) and the solution cooled to 0. DCCI
(0.680 g) was added and the mixture stirred for 20 18 hours before evaporation. The residue was dissolved i in ethyl acetate (50 ml) and washed with aqueous sodium bicarbonate (10~, 2 x 50 ml), water ~2 x 50 ml), dried (Na2S04) and evaporated to dryness. The residue was dissolved in methanol, applied on a column of 25 silica gel ~35 x 2 cm) and eluted with solvent ~5~3~

system lF. ~he fractions containing the desired compound were bulked, evaporated, and the residue dissolved in ethyl acetate (100 ml). The ethyl acetate extracts were washed with aqueous sodium bicarbonate (10~, 3 x 100 ml), dried and evaporated to yield the title compound tetrapeptide (5) as a white solid RflE 0.18, Rf3A 0.39.
~6) H-Tyr-D-Ala-Gly-Mephe-~HcH2cH2cH2NMe2-The protected tetrapeptide (S) (500 mg) was dissolved in ethyl acetate (5 ml) and a solution of hydrogen chloride in ethyl acetate ~5.7 M, 2.5 ml) added to the stirred solution. After 1 hour, the reaction mixture was filtered and the residue washed with petrol ether, dried and lyophilised from water to yield the product (380 mg) as a white solid.
Rf4A 0.27, Rf3B 0 47, ~]2728 ~23.6 ~c = l;
O.lM HCl) EXA.~PLE 3 L-Tyrosyl-DTalanylglycyl-N-methyl-L-phenylalanine-2-dimethylamino~rop~amide-N-oxide l~S~3~

~1) BOC-Tyr(But)-D-Ala-Gly-MePhe-NHCH2CH CH N(O)Me BOC-Tyr(But)-D-Ala-Gly-MePhe-NHCH2CH2CH2NMe2 (500 mg) was dissolved in methanol (10 ml) and an aqueous solution of hydrogen peroxide (30~0, 0.16 ml) added.
The solution was stirred for 48 hours at room ; temperature, filtered and evaporated to yield the desired protected N-oxide as a gum. RflE 0.08;
Rf3A 0.41.
. (2) H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2CH2N(O)Me2_ The gum from (1) above was dissolved in ethyl acetate (5 ml) and a solution of HCl in ethyl acetate (2.63 M, lml) added. The solution was stirred for 1 hour, filtered, the solid dried and lyophilised from water to yield the desired product (240 mg) as a 15 white solid. Rf4A 0.56; Rf3B 0.52 [~ 52728 =+18.8 (c = l; O.lM HCl) EX~MPLE 4 ._ .
L-TyrosYl-D-alanylglycyl-N-methyl-L-phenylalanine-2-dimethylaminoethylamide This was prepared according to the following method:-3~

Tyr D-.~a Gly MePhe ~ BOC ~ H NH2CH2CH2NMe2 But BOC - ~NHCH2CH2NMe2 :BOC--But ~ -OH H---NHCH2CH2~1e22-BOC --/ ~ NHCH2CH2NMe2 H - - ~ HCH2CH2l~Ie22.HCl (1) Boc-~ ephe-~HcH2cH2l\~e2--BOC-MePhe-OH and N,N-dimethylethylenediamine are coupled by the procedure of Example 1(1) to yield the desired amide as an oil.
(2) HMePhe-NHCH2CH2N~e2 ~ c--l The N-protected amide from (1) above was deprotected by the method of Example 1(2) to yield the desired hydrochloride salt as an oil.

(3j BOC-Tyr-(But)-D-Ala-Gly-MePhe-NHCH CH NMe BOC-Tyr- (But) -D-Ala-Gly-OH (7QO mg, 1.5 mM) was dissolved in dry DMF (7.5 ml) and NMM (152 mg~ 1.5 mM) added. The stirred solution was cooled to -15 and IBCF (2~5 mg, 1.5 mM) added. After 5 minutes a solution ~5~30 of 2HCl.H-MePhe-NHCH2CH2NMe2 ~568 mg, 1.5 mM) was added followed by NMM ~303 mg, 3 mM). After 18 hours the reaction mixture was evaporated, and the residue partitioned between EtOAc ~50 ml) and saturated aqueous sodium bicarbonate solution (50 ml). The ethyl acetate layer was separated, washed with saturated aqueous sodium bicarbonate solution (50 ml), water ~2 x 50 ml) and brine (50 ml), dried (Na2S04) and ; evaporated. The residue was chromatographed on silica gel using solvent system 7B. Appropriate fractions were pooled and evaporated to yield the desired tetrapeptide amide (700 mg) as a glassy solid.

(4) 2HCl.H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2NMe2_ The protected peptide from ~3) was deprotected by the method of example 2~6) to yield the desired product. After lyophilsation the tetrapeptide amide ~200 mg) was obtained as a white solid. Rf7B 0.04, Rf3A 0.07, Rf4A 0.23 CY~DO = -24.2 ~c = 0.507DMF) L-Tyrosyl-D-alanylglycyl-L-phenylalanine-thiomorpholineamide-S-oxide ~s~

,'~`
(1) BOC-Phe- ~S
~ BOC-Phe-OH (1.32 g) and IBCF (680 mg) in DME
; (10 ml) were cooled to -20 and NMM (520 mg) was added to the stirred and cooled solution. After 2 minutes a cold suspension of thiamorpholine hydrochloride (690 mg) and N~ (520 mg) in DMF (8 ml) was added.
Stirring was continued for 30 minutes at -20 and then ` at room temperature overnight. After evaporation of ` the solvent the resulting material was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution. The ethyl acetate layer was extracted three times with saturated sodium bicarbonate ~- solution, twice with 10~ solution of citric acid in water, and then with water, and finally with saturated sodium chloride solution. The ethyl acetate solution was dried (Na2S04) and evaporated to give the thiamorpholineamide as a gum (1.56 g).
(2) H-Phe-N S.HCl ~ v --A stirred solution of BOC-Phe-N,,S (1.05 g) in zo acetic acid (3 ml) was treated at room temperature with a solution of hydrogen chloride in acetic aicd (2.6 M, 3 ml), and at two-hourly intervals, with two further 53~0 3 ml portions. After evaporation of the sol~ent and hydrogen chloride, the residue was rubbed with dry diethyl ether, the ether decanted and the residue rubbed with fresh dry ether ~o give phenylalanine thiamorpholine amide as a solid (738 mg).
(3) BOC-Gly-Phe-N S
BOC-Gly-OH (384 mg) was coupled to Phe-N S.HCl (572 mg) by the method used above for BOC-Phe-N~ S, with the use of IBCF (258 mg) and N~l (424 mg total).
774 mg of dipeptide amide was obtained.
(4) BOC-Tyr-(But)-D-Ala-Gly-Phe-N S
BOC-Gly-Phe N~ (697 mg) was stirred with aqueous trifluoroacetic acid (H20 : CF3C02H 1:9 9ml) for lz hours.
The acid was evaporated and the residue was triturated with diethyl ether to give a powder which was dissolved in dry DMF (5 ml) and adjusted with NMM to pH 8 (moist indicator paper). BOC-Tyr-(But)-D-Ala-OH (680 mg) and HONSu (211 mg) in DMF (3 ml) were added followed by DCCI (377 mg) and the mixture stirred overnight at room temperature. The solid was removed by filtration and the filtrate was evaporated to a gum which was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution. The ethyl acetate layer was washed twice with saturated aqueous sodium bicarbonate ~5~

solution then three times with 10% citric acid in water, then with water and saturated sodium chloride solution. Drying ~Na2S04) and evaporation gave the protected tetrapeptide thiamorpholine amide (1.09 g).

(5) Tyr-D-Ala-Gl -Phe-N S
Y ~J
The protected tetrapeptide thiamorpholine amide (1.0 g) was stirred in aqueous trifluoroacetic acid (H20 : CF3C02H 1:9, 9 ml) for 12 hours. Evaporation and trituration with diethyl ether gave a solid which was purified by chromatography on silica gel (CHC13 : CH30H:AcOH: H20 60 : 18 : 1 : 1.5) and then by ion exchange chromatography (Sephadex SP resin, pyridinium form, using a gradient from 2% pyridine in water to 5% pyridine, 1% acetic acid in water). The product was lyophili~ed to give the tetrapeptide - thiomorpholine amide (495 mg).

(6) Tyr-D-Ala-Gly-Phe-~ S-~ O
~,_ Tyr-D-Ala-Gly-Phe-N~S (100 mg) in ethanol (50 ml) was treated with three successive portions of 20 volume hydrogen peroxide (0.3 ml each) at intervals of 45 mins.
with stirring at room temperature. After further stirring for 1 hour the solution was evaporated to dryness, dissolved in water (20 ml) and lyophilised to aive a white solid (9~ mg). Rf3A 0.25; Rf7C 0.06;
~ ~ o ~

ç~ 5330 RfSB 0.38 L~3 15895= f 53.1 (c = 0.47, in 0.1 N HCl).

L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-2-aminoethylamide - 5 Tyr-D-Ala-Gly-MePhe-NHCH2CH2NHZ (prepared by the method of Example 4) (350 mg) was dissolved in MeOH
(20 ml) and added to a suspension of 10% palladium on charcoal (100 mg) in water (10 ml). Hydrogen was bubbled through for 4 hours at 22C. The catalyst was then filtered off and the solvents removed under reduced pressure. The residue was dissolvedin a small amount of 1~ pyridine in water and applied to SP Sephadex resin column (eluted in gradient 1~ aqueous pyridine ~~ 1~ aqueous pyrIdine acetate -~ 5% aqueous pyridine acetate). Relevant franctions were pooled and the solvent removed by evaporation in vacuo.
The product (220 mg~ was freeze dried from water.
Rf3B 0.45 Rf4A 0.56 C~ 2589 = +22.22 (c = 0.98;
0.1 M HCl) EX~MPLE 7 L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine~
3-trimethylarnmoniumpropylamide acetate 5~ ~ V

- 26 ~

(1) BOC-T r-(But)-D-Ala-Gl -MePhe-NHCH CH CH l~ e~OAc~
Y ._ Y ~ -,-, ~
BOC-Tyr(But)-D-Ala-Gly-MePheNHCH2CH2CH2NMe2 (640 mg) was dissolved in ethanol, (5 ml). Methyl iodide (572 mg) was added and the solution stirred at room temperature for 48 hours. The solution was evaporated, the residue dissolved in ethyl acetate and applied to a column of silica gel ~40 x 2 cm) and eluted with ethyl acetate: pyridine:acetic acid: water 90 : 20 : 6 : 11. The -fractions containing the desired compound were combined and evaporated down to afford a solid (320 mg). Rf3A 0.17 Rf3B 0.74 Rf4A 0.47 (2) H-Tyr-D-Ala-Gly-MePhe-NHCH CH CH N~Me Acetate The above protected tetrapeptide (300 mg) was dissolved in ethyl acetate (10 ml) and a solution of HCl in ethyl acetate (3.9 M, 4 ml) added to the stirred solution. After l hour the solution was evaporated and the residue dissolved in water and applied to CM 52 cellulose column (35 x 3 cm) and eluted with linear concentration gradient of 0.05% aqueous pyridine to 1% aqueous pyridine-acetate. The fractions containing the desired compound were combined, evaporated, and the residue lyophilised from water to yield the 33~

~ - 27 -. .
desired product (110 mg) as a white solid. Rf3B 0.17, Rf4A 0.13 Rf5B 0.05 L~ ~589 = ~37'75 (c = 0.5, DMF) L-Tyrosyl-D-methionylglycyl-N-methyl-L-phenylalanine-- 2-dimethylaminoethylamide-N-oxide (1) BOC-Gly-MePhe-NH(CH2)2N(O)Me2_ BOC-Gly-MePhe-NH(CH2)2NMe2 (2.64 g) was dissolved in methanol (200 ml) and meta-chloroperbenzoic acid ~1.23 g) was added. The reaction mixture was stirred - at 22 for 2 hours then allowed to stand at 5 for 18 hours.
' The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate (150 ml) and the solution was washed with saturated aqueous sodium bicarbonate solution (2 x 30 ml), dried ~Na2S04) and evaporated to give the N-oxide (1.9 g) Rf3A 0.76, Rf4A 0.64 ~2) Hcl~H-Gly-Mephe-NH(cH2)2N(o)Me2-The above protected N-oxide (1.8 g) was dissolved in ethyl acetate (10 ml) and acetic acld ~10 ml) was added to the solution. A solution of hydrogen chloride in ethyl acetate ~3.9 ~, 12 ml) was added and the mixture stirred at 21 for 30 minutes.
The solvent was removed in vacuo and the resulting solid triturated with anhydrous diethyl ether ~2 x 40 ml).
The N-oxide hydrochloride (1.5 g) was collected by filtration. Rf4~ 0.40 (3) BOC-Tyr(But)-D-Met-Gly-MePhe-NH(CH2)2N(O)Me2_ BOC-Tyr (But)-D-Met-OH (L.8 g~ was dissolved in dry DMF (10 ml) and HONSu (0.87 g) was added to the solution.
HCl.H-Gly-MePhe-NH(CH2)2N(O)Me2 (1.5 g) and NMM (0.84 ml) were dissolved in dry DMF (20 ml).
The two solutions were mixed, cooled to -10 and DCCI ~0.87 g) was added. The mixture was stirred 15 at -10 for 2 hours then at 20 for 18 hours.
The reaction mixture was filtered and the clear solution evaporated in vacuo. The resulting gum was partitioned between ethyl acetate (60 ml) and water ~10 ml) and the organic phase washed with 10% aqueous 20 sodium bicarbonate solution (1 x 10 ml) and then extracted with 10% aqueous citric acid solution (S x 15 ml).
The pH of the aqueous phase was then adjusted to 7 using solid sodium bicarbonate, and extracted into ethyl acetate (3 x 40 ml). The organic phase was dried 25 ~Na2S04) and evaporated to yield a gum ~1.35 g). Rf3A 0.48 ~3~45330 (4) HCl.H--Tyr-D-Met-Gly-Mephe-NH(cH2)2N(ol!~e2-BOC-Tyr(But)-D-Met-Gly-MePhe-NH(CH2)2N(O)Me2 (1.11 g) was dissolved in a mixture of trifluoroacetic acid (36 ml) and water ~4 ml) containing anisole (0.2 ml). The mixture was stirred for 30 minutes at 22 prior to evaporation in vacuo. The product was purified by silica gel chromatography using a gradient system (solvent systems 3A ~2B) as eluant.
The material from the silica gel column was applied 10 to a carboxymethyl cellulose column eluted with a gradient from 0.1% aqueous pyridine to 1.5%
aqueous pyridine acetate.
Pertinent fractions were pooled, evaporated and the residue freeze dried repeatedly from dilute 15 HCl, to give the desired hydrochloride (341 mg).
Rf3B 0.31 Rf4A 0.44 Rf5B 0.19 C~5899 = +29.30 (c = 0.99, 0.1 N HCl) L-Ty~ y~ methionyl~sulphoxide)glycyl-N-methyl-L
20 phenylalanine 2-dimethylaminoethylamide-N-oxide - HCl.H-Tyr-D-Met-Gly-MePhe-NH(CH2)2N~O)Me2 ~418 mg) was dissolved in ethanol (150 ml) and 100 vol hydrogen peroxide (1.46 ml) was added. The solution was stirred at 21 for 24 hours, after which the solvent was removed under reduced pressure and the resulting material purified by chromatography on CM Cellulose using gradient elution from 0.1% aqueous pyridine to 1.5% aqueous pyridine acetate.
The material from the column was freeze ;dried repeatedly from HCl, to give the desired -~10 hydrochloride (354 mg). Rf3B 0.32 Rf4A 0.43 ;Rf5B 0.32 [~320 ~ ~9.16 (c = 1.O5J 0.1 N HCl) The Table sets out details of further compounds of Formula I prepared by the methods of the above Examples.

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5~30 The following are other typical compounds of the ; invention:-H-Tyr-D-Ala-Gly-MePhe(p-Cl)-NHCH2CH2N(O)Me2.
: H-Tyr-n-Ala-Gly-Phe(p-Cl)-NHCH2CH2N(O)Me2.

CH2CH2CH~
H-Tyr-D-Ala-Gly-MePhe-N
.. \

CH2CH2N (O)Me2 /CH2Ph H-Tyr-D-Ala-Gly-MePhe-N \
" CH2CH2N(O)~Ie2 Ph H-Tyr-D-Ala-Gly-MePhe-N
i,i CH2CH2 N(O)Me2 / CH2CH2 ~ Me H-Tyr-D-Ala-Gly-MePhe-N
CH2CH2N(O)~e2 ,~ ~
CH2CH2 ~ Me H-Tyr-D-Ala-Gly-MePhe-N
CH2CH2N(O)Me2 10 H-Tyr-D-Ala-Gly-MePhe-N l~e CH2CH2N(O)Me2 ~5~3~

/CH2CH2 ~ C
H-Tyr-D-Ala-Gly-MePhe-N

CH2CH2~(0)~e2 CH2 CH2~
H-Tyr-D-Ala-Gly-MePhe-N\ Cl CH2 CH2~3 H-Tyr-D-Ala-Gly-MePhe-N Cl \

CH2CH2N (O)Me2 2CH2 ~ OH
H-Tyr-D-Ala-Gly-MePhe-N

,( CH2CH2N (O)Me2 2 CH2~
OH
H-Tyr-D-Ala-Gly-MePhe-N \-CH2CH2N (O)Me2 H-Tyr-D-Ala-Gly-MePhe-N\ OH
CH2CH2N (O)Me2 j CH2CH2 ~ 3 0Me H-Tyr-D-Ala-Gly-MePhe-N\
CH2CH2N (O)~le2 3~

~Z

H-Tyr-D-Ala-Gly-MePhe-N
OMe CH2CH2N ~O)Me2 ~CH2CH
H-Tyr-D-Ala-Gly-MePhe-N OMe CH2CH2N ~O)Me2 H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2N(O)MeCH2CH2CH3 H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2N(O)MeCH2CH2Ph S H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2NHCONHCH2CH2CH3 ~ -H-Tyr-D-Ala-Gly-Phe-N SO2 H-Tyr-D-Ala-Gly-MePhe-N ~ o H-Tyr-D-Ala-Gly-MePhe-N ~ H

H-Tyr-D-Ala-Gly-Phe-N ~ ~Me CH3C009 ~ CH2CH2Ph Cpm-Tyr-D-Ala-Gly-MePhe-N
CH2CH2N (O)l~.le2 H-Tyr-D-Abu-Gly-MePhe-NHCH2CH2N~O)Me2 H-Tyr-D-Val-Gly-MePhe-NHCH2CH2N(O)Me2 H-Tyr-D-Leu-Gly-MePhe-NHCH2CH2N~O)Me2 H-Tyr-D-Nle-Gly-MePhe-NHCH2CH2N~O)Me2 3~

H-Tyr-D-Thr-Gly-MePhe-NHCHzCH2N~O)Me2 H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2NHMe H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2N(O)MeEt H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2N(O)Et2 H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2NMeEt H-Tyr-D-Ala-Gly-MePhe-NHCH2CH2NEt2.

The starting amines of formula HNR8RX used in the preparation of the above Examples, when not commercially available may be prepared by methods known per se or in the case of particular amines as follows:
i EXAlMPLE A
N-Isoamyl-N',N'-dimethylethylene diamine ; (1) ~le2cHcH2coNH-cH2cH2NH2 Isovaleroyl chloride (5 g, 38 mmole) in dichloro-methane was added dropwise to a stirred solution of N,N-dimethylethylene diamine (3938 g, 38 mmole) and N~IM (7.75 g, 76.7 mmoles) in CH2C12 at 0C. The reaction mixture was stirred at room temperature for four hours when the solvent was evaporated. The residue was taken up in 5_ NaOH (50 ml) and extracted with ethyl acetate (2 x 100 ml). The ethyl acetate was washed with a saturated aqueous solution of sodium chloride, dried (Na2S04) and evaporated. The residue was dissolved in diethyl ether and 5_ HCl in ethyl acetate (10 ml) added.

3~
,~

:
The resulting solid was filtered, washed with diethyl ether (5 x 10 ml) and dried under high vacuum.
Yield = 5.43 g Rf3A 0.40 (2) Me2CHCH2cH2NHcH2cH2N- 2 A solution of the above amide (5 g) in dry THF
` (50 ml) was added dropwise to a stirred suspension of LiAlH4 (2 g) in dry THF (20 ml) at room temperature.
The reaction mixture was then refluxed for 4 hours when t.l.c. showed absence of starting material. A saturated - - 10 aqueous solution of sodium sulphate was added to quench - excess LiAlH4. The resulting suspension was filtered and the filtrate acidified with 4M HCl (10 ml). The solvent was evaporated to leave a white solid which was triturated with diethyl ether, filtered and dried.
Yield - 5.51 g EX~PLE B
, . . _ N-Phenethyl-N',N'-dimeth lethylene diamine (1) Ph CH CONHCH CH NH .HCl This was prepared in a similar manner as described in Example A~l) by coupling phenacetyl chloride ~.0 g, 6.47 mmoles) with N,N-dimethylethylene diamine 0.42 g, 6.47 mmoles) in the presence of N~
Yield = 1.38 g, P~F3A = 0.30 ~2) PhCH2CH2NHcH2cH2NH2 -This was prepared by LiAlH4 reduction of the amide above (10 g) as described for Example A(2). An impure ~ ~5~3~
~ 45 -product ~12 g) was obtained which was purified by silica gel column chromatography eluting with solvent system 3A.
Yield = 4.5 g, Rf3B = 0.31 EXAMPLE C
N-(2-Aminoethyl)-N',N'-dimethylurea ~1) PhcH2ocoNHcH2cH2NHcoNMe2 A solution of dimethylcarbamoyl chloride ~1.39 g) in diethyl ether ~lS ml) was added dropwise to a stirred solution of N~ 1.57 g) and 2-benzyloxycarbonylamino-ethylamine ~3.02 g) in diethyl ether ~110 ml). The mixture was stirred for four hours and allowed to stand for two days. After evaporation of the ether the residual solid was partitioned between 10~ aqueous citric acid and ethyl acetate. The ethyl acetate extracts were I~ashed with 10~ aqueous citric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution, and then dried ~Na2504) and evaporated to give a solid ~1.7 g).
A sample crystallised from ethyl acetate/patroleum ether ~bp 60-80) m.p. 81 - 82 (2) H2NCH2CH2NHCONMe2 The above N-benzyloxycarbonyl compound ~1.06 g) was hydrogenlysed over 10~ Pd/C ~100 mg) in ethanol ~30 ml) containing 2N aqueous hydrochloric acid (2.1 ml) for 12 hours. Filtration and evaporation of the filtrate yielded the desired compound as the hydrochloride which was used without purification.

~ - ` .
~S~30 EX~MPLE D
N-Isoamyl-N-me~hylethylenediamine -(1) BOC-Gly-N(~le)CH2cH2cHrle2 BOC-Gly-OH (17.5 g, 0.1 mole) was dissolved in dry CH2C12 (100 ml) when N~l (10.1 g, 0.1 mole~ and IBCF
(13.7 g, 0.1 mole) were added at -15C. After two minutes N-methylisoamylamine hydrochloride (13.7 g, 0.1 mole) and NMM (10.1 g) were added. The reaction mixture was stirred at -15 C for 1- hours and at room temperature overnight. The solvent was evaporated and the oil dissolved in ethyl acetate, extracted with saturated sodi~m bicarbonate (2 x 100 ml), 10% aqueous citric acid (2 x 100 ml) washed with water until neutral, dried (Na2S04) and evaporated. An oil was obtained.
Yield = 27g (quantilative) Rf7D = 0.79 (2) -2NCH2cH2N(Me?cH2CH2CHMe2 BOC-Gly-N~Me)CH2CH2CHMe2 ~12 g) was dissolved in 90~ TFA aqueous trifluoroace-tic acid ~H20 : CF~COOH
1:9, 35 ml). After two minutes the solvent was evaporated to leave an oil. The oil was dissolved in 50 ml of freshly dried THF and added to diborane in THF ~lM, 150 ml) under an atmosphere of nitrogen at -20C. The temperature was allowed to reach room temperature and the reaction mixture stirred overnight. Methanol~100 ml) was carefully added to destroy the excess diborane and solvent evaporated to ~45~33V

yield an oil~ The oil was then treated with 6 ~I HCl ~50ml). The reaction mixture was refluxed for 3 hours and evaporated down to an oil. This oil was further treated with 2M NaOH for 1 hour, acidified and evaporated. The oil so obtained was purified by silica gel column chromatography (2.5 cm x 80 cm) with solvent system 3A to yield the desired diamine.
Yield = 4.4 g, Rf3A = 0.20 EX~MPLE E
. .
Dimethyl(2-amino-4-methylpentyl)amine (1) BOC-Leu-NMe2 BOC-Leu-OH (4.62 g, 20 mlM) was dissolved in CH2C12 and the solwtion cooled to -20. N~ (2.25 ml, 20 mM) and chlorodiphenylphosphate oxide (6.0 g, 20 m~) were added, followed after 15 minutes by dimethylamine (5.6 g, 125 mM). After 3 hours at 20 the solution was evaporated and the residue partitioned between ethyl acetate and water (50 mls each). The organic phase was washed with 10~ aqueous citric acid~ aqueous sodium bicarbonate and saturated brine, dried (Na2S04) and evaporated to yield the desired amide (5.15 g).

e2NCH2CH ( .N~2 ) CH2CHMe2 BOC-Leu-NMe2 (1.3 g, 5 mM) was dissolved in aqueous trifluoroacetic acid (H20 : CF3COOH 9:1, 20 ml) and the solution stirred for lhour. The solvent was then evaporated and the residue dissolved in dry THF. The stirred solution was cooled to -20 under nitrogen and a solution of diborane in THF (1 ~I, 30 ml) added. After S hours at room temperature, methanol (10 ml) was added and the solution stirred for 12 hours.before ~` S evaporation. The residue was dissolved in aqueous HCl ~` (2M, 15 ml) and stirred for a further 12 hours before evaporation. The residue was purified by chromatography in silica gel (system 7C ~7B). Pertinent fractions were pooled and evaporated to yield the desired diamine as an oll (800 mp) which crystallised on standing.

r - ~
1~53;~0 :

.

`~ In the above mentioned test method of ~osterlitz et al male or female guinea pigs (Duncan Hartley strain) are killed by a blow on the head and a portion ~ of the ileum removed and set up in an isolated - 5 organ bath of 50 ml volume. A 'twitch' response is produced by low frequency (0.1 Hz) stimulation with 0.5 msec rectilinear pulses. A test compound is dissolved in distilled water to produce a stock solution of concentration 1 mg/ml. Serial dilutions are carried out using Krebs solution to produce concentrations of 10 ,ug, l,ug and O.l~ug/ml. The compound is tested by adding between 0.1-0.3 ml of the solutions to the organ bath. A dose response curve is then drawn and compared with that for Met enkephalin.
In the rat tail flick test hot water (maintained at 55-1C) is used as the nociceptive stimulus.
The following are data obtained with compounds of the invention when tested by the guinea pig ileum tG.P.I., activity compared with Met enkephalin), Hendershot et al ( H & F) and rat tail pressure (R.T.F) tests.

~S3~0 - so -.
, . , Example G.P.I. Poténcy H & F ED50 R.T.F. E~50 No. (~ Met enk~halin~ mg/Kg i.v. mg/K~ i.v.
1 2.6 0.01 2 7.2 0.28 2.0 3 4.1 0.000.03 1.8 4 8.5 0.003 1.4 --22 6.5 0.08 ng/Kg 0.32 - 6 13.0 0.028 0.37 lg 23.2 0.24 ~o~hine~ 1.0 0.07 2.2`

The pharmaceutical compositions may be, for example, in a form suitable for parenteral administration such as sterile injectable aqueous or oily solutions or suspensions.
The parenteral dosage forms suitable for intraveneous 15 intramuscular or subcutaneous administration contain from 0.1 to S0 mg/ml of a compound of the invention (or an equivalent amount of a pharmaceutically acceptable salt).
Dose levels will generally, for the relief of pain, be in the range of 0.1 to 100 mg depending upon the route of 20injection.

,

Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN
EXCLUSIVE PROPERTY OF PRIVELEGE IS CLAIMED ARE
DEFINED AS FOLLOWS:

1. A process for the preparation of a novel peptide compound of the formula I

wherein R1 is hydrogen or methyl;
B is D-alanine, D-valine, D-norvaline, D-serine, D-methionine or D-methiohine sulphoxide, D is L-phenylalanine or N-methyl-L-phenylalanine;
R8 is hydrogen, alkyl C1-5, Ar or Ar-alkyl C1-2;
Rx is a group (CH2)s - X
where s is 2-4 and X is NHR9, N?R11R12R13, NR9COR11, NR9COOR11, NR9CONH2, NR9CONR10R11, N(O)nR11R12, COOR9, or OCONR9R10 (where R9 is hydrogen, alkyl C1-3, Ar or ArCH2; R10 is hydrogen or alkyl C1-3; R11 is alkyl C1-3, hr or ArCH2; R12 is alkyl C1-3; R13 is alkyl C1-3;
and n is 0 or 1);
or the group is where M is 1-2 and Q is SO or N(O)R11;
Ar is phenyl or phenyl substituted by chlorine, methyl, hydroxy or methoxy; which process is characterised in that a compound of formula Y Ml OH II
wherein Y is a suitable N-protecting group and M1 is a suitably protected amino acid or peptide residue, is coupled with a compound of formula III
wherein Rx and R8 are as hereinbefore defined and M2 is a suitably protected amino acid or peptide residue (where M1 and M2 together represent R1-Tyr-B-Gly-D or a protected derivative thereof and where R1, B and D are as hereinbefore defined), followed by removal of the protection and thereafter if desired when Rx is (CH2)s-N(O)nR11R12 (where n is O) oxidising with hydrogen peroxide or metachloroperbenzoic acid to produce the compound where Rx is (CH2)sN(O)nR11R12 (where n is 1).

2. Compounds of formula I as defined in claim 1.
whenever prepared by the process of claim 1.

3. A process for the preparation of a novel peptide compound of the formula:
I

wherein R1 is hydrogen or methyl;
B is D-alanine, D-valine, D-norvaline, D-serine, D-methionine or D-methionine sulphoxide;
is L-phenylalanine or N-methyl-L-phenylalanine;
R8 is hydrogen, alkyl C1-5, Ar or Ar-alkyl C1-2;
Rx is a group (CH2)s - X
where s is 2-4 and X is NHR9, NR9COR11, NR9CONH2, NR9CONR10R11, N(O)nR11R12, COOR9, CONR9R10, NR9COOR11 or OCONR9R10 (where R9 is hydrogen or alkyl C1-3; R10 is hydrogen or alkyl C1-3; R11 is alkyl C1-3; R12 is alkyl C1-3 and n is 0 or 1);
Ar is phenyl or phenyl substituted by chlorine, methyl, hydroxy or methoxy; which process is characterised in that a compound of formula wherein Y is a suitable N-protecting group and M1 is a suitably protected amino acid or peptide residue, is coupled with a compound of formula wherein Rx and R8 are as hereinbefore defined and M2 is a suitably protected amino acid or peptide residue (where M1 and M2 together represent R1-Tyr-B-Gly-D or a protected derivative thereof and where R1, B and D are as hereinbefore defined), followed by removal of the protection and thereafter if desired when Rx is (CH2)s-N(O)nR11R1 (where n is 0) oxidising with hydrogen peroxide or metachloroperbenzoic acid to produce the compound where Rx is (CH2)sN(O)nR11R12 (where n is 1).

4. Compounds of formula I as defined in claim 3 whenever prepared by the process of claim 3.

5. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is L-phenylalanine, Rx is -CH2CH2NHCOCH3 and each of R1 and R8 is hydrogen.

6. L-Tyrosyl-D-alanylglycyl-L-phenylalanine-2-acetamidoethylamide whenever prepared by the process of claim 5.

7. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phenylalanine, Rx is -(CH2)3N(CH3)2 and each of R1 and R8 is hydrogen.

8. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-3-dimethylaminopropylamide whenever prepared by the process of claim 7.

9. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phenylalanine, Rx is -(CH2)3N(O)(CH3)2 and each of R1 and R8 is hydrogen.

10. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-3-dimethylaminopropylamide-N-oxide whenever prepared by the process of claim 9.

11. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phenylalanine, Rx is -(CH2)2N(CH3)2 and each of R1 and R8 is hydrogen.

12. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-2-dimethylaminoethylamide whenever prepared by the process of claim 11.

13. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phenylalanine, Rx is -(CH2)2N(O)(CH3)2 and each of R1 and R8 is hydrogen.

14. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-2-dimethylaminoethylamide-N-oxide whenever prepared by the process of claim 13.

15. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phenylalanine, Rx is -(CH2)2NH2 and each of R1 and R8 is hydrogen.

16. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-2-aminoethylamicle whenever prepared by the process of claim 15.

17. A process according to claim 1 wherein in the compound of formula I so obtained B is D-alanine, D is N-methyl-L-phcnylalanine, Rx is -(CH2)3CON(CH3)C6H5 and each of R1 and R8 is hydrogen.

18. L-Tyrosyl-D-alanylglycyl-N-methyl-L-phenylalanine-y-aminobutyric-N-methyl-N-phenylamide whenever prepared by the process of claim 17.